Scholarly article on topic 'Efficiency of Microalgae Chlamydomonas on the Removal of Pollutants from Palm Oil Mill Effluent (POME)'

Efficiency of Microalgae Chlamydomonas on the Removal of Pollutants from Palm Oil Mill Effluent (POME) Academic research paper on "Agriculture, forestry, and fisheries"

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{"Nutrient removal" / "Chlamydomonas incerta" / Microalgae / "Palm Oil Mill Effluent"}

Abstract of research paper on Agriculture, forestry, and fisheries, author of scientific article — Hesam Kamyab, Mohd Fadhil Md Din, Ali Keyvanfar, Muhd Zaimi Abd Majid, Amirreza Talaiekhozani, et al.

Abstract Malaysia is considered as a major palm oil producer in the world. Therefore, it is vital to utilize an environmentally friendly and inexpensive method to treat palm oil mill effluent (POME) in Malaysia. Nowadays, the use of microalgae to remove pollutants from POME has gained a lot of attention. The main objective of this research was to investigate the effect of POME as a nutrient on the microalgae growth and analyze the removal rate of pollution. In this study, a pure culture Chlamydomonas incerta was aseptically transferred to an Erlenmeyer flask containing POME. The effect of POME as a high nutritional substrate, different cultivation scales, carbon total nitrogen (C:TN) ratio, and the lipid productivity of microalgae C. incerta were assessed. C. incerta was grown at room temperature under continuous illumination with the intensity of ± 15 (μmol/m2/s) for 28 days, followed by the measurement of chemical oxygen demand (COD) reduction at different substrate concentrations. The results of this study demonstrated that organic carbon was removed by C. incerta for the ratio of 100:7, 100:13, and 100:31 respectively within the second day of cultivation. Fast growth of microalgae was observed in organic and inorganic substrates for adoption within the second day of experiment. The optimum achievement rate of nutrient removal with C. incerta was about 67.35% of COD for 250mg/L of POME concentrations in 28 days. The significance of this study is regarding the introduction of a new microalgae strain with a high ability to remove nutrients from POME, which can contribute to the effort in finding an efficient and economic technology for improving our environment.

Academic research paper on topic "Efficiency of Microalgae Chlamydomonas on the Removal of Pollutants from Palm Oil Mill Effluent (POME)"

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Energy Procedía 75 (2015) 2400 - 2408

The 7th International Conference on Applied Energy - ICAE2015

Efficiency of Microalgae Chlamydomonas on the Removal of Pollutants from Palm Oil Mill Effluent (POME)

Hesam Kamyaba, Mohd Fadhil Md Dina, Ali Keyvanfarb, Muhd Zaimi Abd Majidb*, Amirreza Talaiekhozania, Arezou Shafaghatb, Chew Tin Leec, Lim Jeng

Shiunc ,Hasrul Haidar Ismailb

aInstitute of Environmental and Water Resource Management (IPASA), Faculty of Civil Engineering, Universiti Teknologi Malaysia, Johor, 81310, Malaysia bConstruction Research Centre (CRC), Construction Research Alliance (CRA) , Faculty of Civil Engineering, Universiti Teknologi

Malaysia, Johor, 81310, Malaysia c Department of Bioprocess Engineering, Faculty of Chemical Engineering, Universiti Teknologi Malaysia, Johor, 81310, Malaysia

Abstract

Malaysia is considered as a major palm oil producer in the world. Therefore, it is vital to utilize an environmentally friendly and inexpensive method to treat palm oil mill effluent (POME) in Malaysia. Nowadays, the use of microalgae to remove pollutants from POME has gained a lot of attention. The main objective of this research was to investigate the effect of POME as a nutrient on the microalgae growth and analyze the removal rate of pollution. In this study, a pure culture Chlamydomonas incerta was aseptically transferred to an Erlenmeyer flask containing POME. The effect of POME as a high nutritional substrate, different cultivation scales, carbon total nitrogen (C:TN) ratio, and the lipid productivity of microalgae C. incerta were assessed. C. incerta was grown at room temperature under continuous illumination with the intensity of ± 15 (^mol/m2/s) for 28 days, followed by the measurement of chemical oxygen demand (COD) reduction at different substrate concentrations. The results of this study demonstrated that organic carbon was removed by C. incerta for the ratio of 100:7, 100:13, and 100:31 respectively within the second day of cultivation. Fast growth of microalgae was observed in organic and inorganic substrates for adoption within the second day of experiment. The optimum achievement rate of nutrient removal with C. incerta was about 67.35% of COD for 250 mg/L of POME concentrations in 28 days. The significance of this study is regarding the introduction of a new microalgae strain with a high ability to remove nutrients from POME, which can contribute to the effort in finding an efficient and economic technology for improving our environment.

©2015Published byElsevierLtd.Thisisanopen access article under the CC BY-NC-ND license

(http://creativecommons.Org/licenses/by-nc-nd/4.0/).

Peer-review under responsibility of Applied Energy Innovation Institute

Keywords: Nutrient removal; Chlamydomonas incerta; Microalgae; Palm Oil Mill Effluent

* Corresponding author. Tel.: +6-075533107; fax: +6-075533114. E-mail address: mzaimi@utm.my.

1876-6102 © 2015 Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license

(http://creativecommons.Org/licenses/by-nc-nd/4.0/).

Peer-review under responsibility of Applied Energy Innovation Institute

doi: 10.1016/j .egypro .2015.07. 190

1. Introduction

The Malaysian palm oil industry has grown rapidly over the years, which makes Malaysia as the world's largest producer and exporter of palm oil and its derivatives. Palm oil sludge or palm oil mill effluent (POME) is a wastewater generated by processing oil palm and consists of various suspended materials [1]. POME has a very high biochemical oxygen demand (BOD) and chemical oxygen demand (COD), which is 100 times higher than municipal sewage. The effluent also contains high concentration of organic nitrogen, phosphorus, and other nutrient contents [2]. POME is a colloidal suspension consisting of 95-96%water, 0.6-0.7% oil and 4-5% total solids, including 2-4% suspended solids originating from a mixture of sterilizer condensate, separator sludge and hydrocyclone wastewater [3]. Microalgae are microscopic photosynthetic organisms that can be found in both marine and freshwater environments. There are many varieties of microalgae and each species has a different proportion of lipids (fats), starch, and proteins. [4]. Microalgae capture light energy and utilize it for cell synthesis and proselyte inorganic molecules into simpler sugar as a source of energy for cell metabolism [5]. Nitrogen and phosphorus are two necessary and fundamental components for the growth of the algae, which are rich in POME wastewater [6]. The use of microalgae has gained attention of researchers due to its ability to remove a wide range of pollutants from water such as nitrogen and phosphorus [7]. Agricultural wastewater such as POME can be treated by using algae [8]. Releasing POME without sufficient treatment into aquatic environments can increase both biological oxygen demand (BOD) and chemical oxygen demand (COD). Microalgae have been used for wastewater treatment and biomass production since 1950 [9]. It implies that Chlamydomonas could be a decent agent to remove heavy metals and other toxic components from wastewater released into the natural environment. Nitrogen and phosphorus are two important pollutants in POME that can severely polluted aquatic environments. Several studies have demonstrated that microalgae under particular conditions can be used to expel nitrogen and phosphorus from POME wastewater [10]. Microalgae can naturally exist in lagoons containing POME. Regrettably in Malaysia, a huge amount of POME is discharged daily without complete treatment into natural water resources such as rivers, which causes colossal negative ecological effect. The conventional treatment methods (e.g. anaerobic digesters and aerated lagoons) are unable to meet the regulations set by the Department of Environment (DOE). According to DOE, the level of BOD for wastewater to be released into aquatic environments must be less than 100 mg/L [1, 11]. Nevertheless, algae can grow in POME even in the presence of heavy metals. Therefore, microalgae can be used for removal of organic compounds and heavy metals simultaneously [12, 13]. In addition, microalgae can absorb carbon dioxide from the atmosphere and release oxygen in water, which will help to concentrate more oxygen to the system. Moreover, the widespread use of microalgae leads to a solution for decreasing carbon dioxide in the atmosphere [14, 15]. Recent studies have shown that microalgae can be used for biofuel production. It means cultured microalgae for POME treatment can be used for biofuel production. Research demonstrates that microalgae contain less water than terrestrial plants and as the cells are grown in suspension, thus they have higher efficiency of access to water [16, 17 and 18]. For the same reason, they perform well in taking up CO2 from different supplements.

Although much research has been carried out to remove phosphorous and nitrogen from POME by using microalgae, no one could find any highly efficient microalgae strain to treat POME. Among all microalgae that naturally live in POME, Chlamydomonas has faster growth and high ability to remove pollutants from POME. The aim of this study was to evaluate the ability of Chlamydomonas for removal of nitrogen and phosphorous from wastewater polluted with POME. In this study, Chlamydomonas was isolated from pond containing POME and was assessed for its ability to remove different pollutants including phosphorus, nitrogen and COD. Overall, the results of this study are important to introduce

microalgae as an option for treatment to prevent environmental pollution by industries generally and by palm oil industry particularly.

2. Materials and Method

The cultivation of microalgae C. incerta was performed with diverse concentrations of POME at 0, 250, 500 and 1000 (mg COD/L), and the growth rate was determined using chemical oxygen demand (COD) measurements. Furthermore, analytical measurements were performed based on the standard methods for the examination of water and wastewater. Moreover, modified Bold's basal medium (BBM) was utilized to enhance the growth of microalgae in POME [19].

2.1. Preparation of Inoculum

The research was conducted by selecting C. incerta, which was isolated and well grown in IPASA-UTM Laboratory, Malaysia as commercial species of green microalgae. The microalgae were cultured and adopted in the appropriate synthetic medium (BBM) as its natural habitat and appropriate in the tropical region [4].

2.2. Wastewater Collection

The carbon source and mixed consortia of microalgae were obtained from facultative ponds at FELDA palm oil mill in Kulai, Johor, Malaysia, and the specimen obtained was stored in 5 L plastic containers with proper labelling. The period of sampling was every three months, followed by freezing at about 4°C to prevent any contamination and to limit the activity of biodegradation process. Prior to sample preparation for microalgae cultivation, collected POME was left over in order to return the POME to normal room temperature. The sludge and other hazardous materials present in POME after sedimentation and filtration were sufficiently separated for 1 h. POME was diluted ten times to decrease the shading effects on the growth of microalgae [4]. Furthermore, the particulars of POME utilized as a part of this study are indicated in Table 1.

Table 1. Characteristics of the POME sample

No Parameter * Concentration (mg /L) Average (mg/ L)

1 pH 4.15 - 4.4.5 4.25

2 COD 1,350 - 2,120 1,600

3 Soluble COD 20,500 - 24,500 22,000

4 BOD 300 - 400 330

5 Total volatile solid 27,300 - 30,150 28,100

6 Total suspended solid 15,660 - 23,560 18,900

7 Total phosphorus 200 - 600 350

8 Total nitrogen 500 - 800 500

*All parameters are in units of mg/L except pH (Source: Kamyab et al., 2014)

2.3. Antibiotic Preparation

Antibiotic chloramphenicol with 99% purity was used throughout this study. Chloramphenicol dissolved in ethanol was prepared as a supporting solution for the sterilization stage in order to reduce undesirable particles, which affect medium preparation. At that point, syringe with a diameter of 0.2 mm

filter membrane was utilized to sterilize chloramphenicol solution. After disinfection, the antibiotic was added into the synthetic medium to achieve final concentration of 30 ^g/mL. The antibiotic stock solution was sealed with parafilm and stored at 4°C until further use.

2.4. Chemical Oxygen Demand (COD)

The standard protocol for COD measurement was used to determine the COD levels displayed in the sample. It was performed based on the previous method of water and wastewater examinations [20]. The purpose of this test is to estimate the potential of algae in reducing the COD substance from wastewater. The algal cells were completely homogenized using vortex that was obtained after being included into COD vials and afterward, the vials were mixed well and placed in a preheated digester block at 1,500°C for 2 h. At that point, the samples were mixed and measured using the 5220 B method [20].

2.5. Total Nitrogen Measurements

2 mL microalgae suspension was collected from each Erlenmeyer flask for the investigation of nutrient consumption starting from the inoculation period. The samples were initially centrifuged at 4,000 rpm for 10 min and afterward, the supernatants were legitimately diluted and analyzed for the total nitrogen based on the HACH DR 5000 spectrophotometer manual [11].

2.6. Experimental Procedures

C. incerta was inoculated (2%, v/v) in a liquid medium. Heterotrophic cultivation of C. incerta was initially carried out using a 250 mL Erlenmeyer flask containing 100 mL of a liquid medium. Furthermore, POME used in batch experiments was settled and further diluted in BBM at respective COD concentrations of 250, 500 and 1,000 mg/L. Additionally, controlled sample without POME (0 mg/L) was prepared for the experiment. C. incerta was grown at room temperature under continuous illumination with the intensity of ± 15 (^mol/m2/s) for 28 days, followed by the measurement of chemical oxygen demand (COD) reduction at different substrate concentrations.

Then, POME and synthetic medium were mixed together to support the growth of microalgae. POME with the concentration of 250 mg COD/L, synthetic medium and 10% (v/v) of microalgae were added together in the media and kept at ambient room temperature (30°C) under continuous illumination within the intensity of ± 15 (^mol/m2/s) for 20 days. Finally, 1 mL of antibiotic solution (chloramphenicol) was added to the medium in order to verify that the culture would be free of contamination.

2.7. Inhibition Model

In order to determine the characteristics of microbial growth in diverse initial concentrations of POME and to examine the impact of different substrate concentrations on the quality of microalgae cultivation, a kinetic model was applied to the acquired test results. In this case, an inhibition model was used as a suitable model to fit the data identified with the conduct of C. incerta during the experiment. The most well-known inhibition models used for describing cell growth are Haldane, Linear, Exponential and Teissier models, which are removed from modified-Monod equations used for substrate inhibition in enzymatic reactions [21, 22, 23]. As indicated by this model, the microbial growth can be determined through the following equation:

Ks +[S +

where S is the substrate concentration, is the specific growth rate of the microorganisms, ^m is the maximum specific growth rate, Ks is the half immersion steady for microbial growth and, and Ki is the separation constant for substrate inhibition with distinctive concentrations. Estimations of bio-kinetic constant Ks and Ki were acquired using Graph Pad Prism Software (evaluation).

3. Results and Discussion

3.1. Effect of Palm Oil Mill Effluent (POME) as substrate

COD values in Fig. 1 decreased slightly during the experiment, with the lowest value achieved at the end of the experiment. It means C. incerta uptakes the COD as a substrate for reserve by itself. The COD values diminished with time for the diverse concentrations of POME as demonstrated in Fig. 1, and the results represent that C. incerta removed about 68.24%, 67.35%, 43.20%, and 34.12% of COD for 0 mg/L, 250 mg/L, 500 mg/L and 1,000 mg/L of POME concentrations, respectively. Furthermore, the highest value was achieved by 0 mg/L as a control without using of POME. As C. incerta is able to achieve optimum growth in the absence of POME, this indicates that POME may have an inhibitory effect towards the growth of C. incerta. It can be concluded that C. incerta is considered as the compelling species for the elimination of natural substances at the POME concentration around 250 mg/L, while it reacted quite slow in growth when the concentration of POME was raised to 500 and/or 1000 mg/L. As an alternate correlative test, when C. incerta was grown in batch culture with piggery wastewater, the organic substance removal by C. incerta was recorded at 88%, 55.6% and 20.6% for the COD concentrations of 250 mg/L, 520 mg/L and 1,100 mg/L, respectively [23].

COD of POME (mg/l(

Fig. 1. Ability of C. incerta to remove pollutants from POME in different COD at 28 days

3.2. Substrate Consumption Rate

Fig. 2 displays a slight abatement in the utilization of substrate during experiment, where the most reduced value was accomplished towards the end of the investigation. It implies that C. incerta uptakes the COD and utilize it as a substrate for reserve by itself. It was clear that COD consumption rate was just about 58% following 28 days of cultivation. COD removal and consumption rate most elevated values were attained by the substrate without POME (0 mg/L). This means that C. incerta could be ready to attain the ideal growth without POME. It shows that POME may have an inhibitory impact towards the growth of C. incerta. Fig. 3 presents higher growth rate for the cells at 250 mg/L of POME, a lower growth rate at 1,000 mg/L and intermediate growth rate was observed at 500 mg/L concentration of POME. Likewise, the logarithmic growth proportions of (XTX'0) versus operational time for different substrate concentrations are illustrated in Fig. 3, where X' represents the growth of microalgae (absorbance) during experiment, and X'0 signifies the value of growth (absorbance) towards the start of the experiment.

1000 900 800 700 600 500 -400 300 200 -100 0

0 (mg/L) 250 (mg/L) 500 (mg/L) 1000 (mg/L)

6 10 14 16

Time (days)

1.80 1.60 1.40 1.20 1.00 0.80 0.60 0.40 0.20 0.00

0 (mg/L) 250 (mg/L)

500 (mg/L)

1000 (mg/L)

2 6 10 14 Time (days)

16 20 28

Fig. 2. Substrate consumption versus operation time for

C. incerta

Fig. 3. Growth rates of microalgae under different initial substrate concentrations for C. incerta

3.3 Inhabitation rate

Fig. 4 presents experimental and model results for a particular growth rate as a function of initial POME concentration. The estimations of specific growth rates were set for each initial POME concentration (S) by plotting ln (X/Xo) versus time. This information was then fitted to equation by nonlinear least-squares regression techniques for estimation of the biokinetic constant. The critical concentration of POME was 1,000 mg/L for all measurements, including Optical density (OD), Mixed liquor suspended solids (MLSS) andMixed liquor volatile suspended solids MLVSS. The results show that specific growth rate decreased with increasing substrate concentration. It implies that substrates at higher concentrations act as inhibitors for microalgae growth. The results obtained are comparable to those obtained by other authors using piggery wastewater as a medium for the growth of microalgae [23].

0.005 0.004 0.003 0.002 0.001

0.000 0.200 0.400 0.600 0.800 1.000 1.200

0.005 0.004 0.003 0.002 0.001

Experiment Model

0.000 0.200 0.400 0.600 0.800 1.000 1.200

0.007 0.006

0.005 0.004 0.003 0.002 0.001

0.000 0.200 0.400 0.600 0.800 1.000 1.200

Fig. 4. Comparison of the model values and experimental data; A (MLSS measurements), B (MLVSS measurements), C (OD

measurements) for C. incerta performance

3.4. Organic Carbon Substrate and Nutrient Utilization Rate

Fig. 5 demonstrates the rate for natural carbon and supplement use with time for three separate carbon to total nitrogen proportions (100:7; 100:13; 100:32). All the proportions achieved a noteworthy rate within two days of cultivation, proposing that microalgae did not need a long time adapting to organic and inorganic compounds for growth. After two days, the carbon source (substrate) diminished gradually, which showed that the microalgae used carbon source not only for growth, but also for lipid production as noticed beforehand. This result repudiates with Fig. 6, whereas C. incerta consumed carbon source rapidly within 7 days. Meanwhile, at this stage, the microalgae had attained to fitting supplement (e.g. nitrogen), so it might grow successfully. Organic carbon had definitely expanded within the first two days of microalgae cultivation, and dropped gradually until the end of experiment (see Fig. 5). Before the end of the experiment, 56.2%, 54.7%, and 49.8% of organic carbon was removed by C. incerta for the ratio 100:7; 100:13; and 100:31, respectively. It implies that the algae species could use distinctive organic compounds as carbon sources [24]. Moreover, the microalgae species utilized as a part of study incorporated the mixotrophic nature, which could use light and natural source as the energy source simultaneously [24,25].

J 50 =

Carbon substrate rate for C/TN=100:7 Carbon substrate rate for C/TN=100:13

Time(days)

Chlorella vulgaris Chlorella sorokinia^ . ^ A......................

..........x..........X...........x...........X-

Time(days) 15

E 2.00

^ 1.50

Fig. 5. Organic carbon substrate rates

Fig. 6. Lipid content in five strains of microalgae in batch culture

4. Conclusion

The results demonstrated that the highest removal rate of nutrients in 250 mg/L of POME with microalgae Chlamydomon incerta was about 67.35% of COD concentration. Based on the findings, organic carbon with the ratios of 100:7, 100:13, and 100:31 was removed by C. incerta within the second day of cultivation. In addition, C. incerta was able to reach relatively optimum lipid content and lipid productivity when cultured in POME with C: TN ratio at 100:7. The research was conducted at constant temperature and photoperiod. It is proven that nitrogen varieties could play a crucial role in lipid accumulation in microalgal cells. The consequences of this study exhibit that C. incerta can be a suitable species to remove hazardous components and supplements exist in POME. Cultivation of microalgae in wastewater ponds is easy, straightforward operation and environmental friendly. Hence, it can be utilized as practical operators to treat wastewater released from different industries. The procedure of microalgae cultivation in POME along with efficient treatment reduces cost, energy and greenhouse gas emission. For future research, C. incerta may be considered as an applicable microalgae species for treating other sources of wastewater because microalgae can be easily grown in POME and is abundantly present throughout the year in Malaysia.

Acknowledgments

The authors would like to acknowledge CRC and IP ASA, Universiti Teknologi Malaysia for providing adequate facilities to conduct this research and for their financial support of this research through the Research University Grant and MOSTI fund with Vot No. 4S042.

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Biography

Prof. Dr. Muhd. Zaimi Abd. Majid is a senior researcher serving as the Dean of Construction Research Alliance, Universiti Teknologi Malaysia. His expertise is in sustainable construction management research area, in which he has established since the late 1980s. His Ph.D and Masters in Construction Management was completed in 1997 from Loughborough University. He has more than 100 publications in international peer-reviewed journals with hundreds of cumulative citations. He has experience as the guest editor, editorial board and board of reviewers of several impact factor journals as well. In addition, he has been leading in multidisciplinary national and international research projects and has received gold awards for several international research projects in different fields throughout his career. Finally, he has won several awards, including gold and jury award for research products.